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Care should be taken to use either glass or polypropylene (depending on the hormone; see Table 8.2) beakers, tubes, and pipettes for solubilizing, diluting, and aliquoting factors to minimize loss due to sticking. At best, one only knows what concentration of hormone one starts with, not what concentration is delivered to a culture, much less what remains after 1 hr, 1 day, or at the end of the experiment. This can only be ascertained by a direct measurement of hormone levels in the medium or cells. (This, however, is seldom done.)
A few words should be said about the source and quality of hormones. Except for synthesized or recombinant hormones, most are "hormone preparations." These will vary from one supplier to another and from one lot to another in activity and purity. It is possible that
the growth-promoting activity in factor X is actually factor Y, present as a minor contaminant. If the purpose of an experiment is to study the effect of, for instance, follicle-stimulating hormone (FSH), the National Institutes of Health (NIH) FSH (or other FSH source) may be used; however, one will eventually need to show, as far as possible, that it is the FSH and not contaminating luteinizing hormone (LH), growth hormone (GH), thyrotropin (TSH), and so on that are eliciting the observed "FSH" effects. This can be done by using blocking antibodies, if possible, or comparing very highly purified hormone preparations from several sources, including recombinant.
Many suppliers will provide samples of several batches of a hormone for testing. One can then purchase a large amount of the lot that is the most effective and use the same lot in the series of experiments. If dose is critical, dose'Cresponse curves should be performed on each new lot of hormone. It is wise to maintain a skeptical attitude toward manufacturers' and suppliers' claims of activity and purity of hormones, particularly those purified from pituitary or sera that contain many active components. Manufacturers may also add buffers, BSA, or some other protein carrier to hormone preparations to act as a stabilizer. If so, you should have an excipient-only control. If you have the facilities, it is best to verify purity levels. Simple sodium dodecyl sulfate"Cpolyacrylamide electrophoresis (SDS-PAGE) of the preparations can be an informative and sometimes a surprising and depressing experience. However, there is now usually a choice of suppliers for all of the commonly used attachment and growth factors. It is thus possible to choose the best source as to price, purity, and so forth.
Finally, it should be kept in mind that some hormones and growth factors (especially peptide hormones) show a species specificity for binding and/or activity. While it is frequently not possible to use hormones isolated from the homologous species, comparisons of the effect of hormones from the homologous species should be performed where possible. Heterologous hormones may be equally effective or effective at higher concentrations but much less expensive. Even in those cases where hormones' structures are identical, the active form may vary from species to species or from one tissue to another.
As an example of the points discussed above, Fig. 8.6 shows a growth-promoting effect of an impure FSH preparation on RL-65 cells that could be reproduced by pituitary extract but not by purified FSH. Obviously, in this case, FSH is not the true mitogen but rather another pituitary factor in the FSH preparation. Neutralizing antibodies or receptor antagonists, if available, can also be used to prove that the major constituent of a hormone preparation is truly the active factor. It is also desirable where possible to have a positive control. Thus, if the fact that cell "X" does not respond to FGF is important to the hypothesis, the FGF preparation should be shown to be active on a cell line known to respond.
Subculture and Setting Up Experiments
Most cell lines are routinely carried in a serum-supplemented medium. Since these established cell lines have been grown in serum-supplemented medium for a period of years, it can be assumed that to a greater or lesser extent the serum supplement is providing the cells with the hormones required for growth and survival in vivo and/or the cells have adapted to the absence or reduced levels of some of the hormones required. The absence of sufficiently elevated levels of some hormones in 5"C20% serum may in part account for the difficulty in establishing some cell types in culture. This point will be discussed in the following chapters on primary cultures and establishing cell lines.
Experiments on deriving serum-free medium to replace the requirement for serum are initially performed using stock plates that have been grown in serum-containing medium. This is to avoid selecting for cells with reduced hormone requirements. However, selecting for cells that do not require serum is a valid approach if the desired endpoint is to obtain a cell that requires a minimum of addition to the nutrient medium for growth. This approach will not help one understand what it is in serum that is required for the cells' growth nor will it aid in understanding the in vivo requirements of the cell.